Single-channel multiplexing without melting curve analysis in real-time PCR

Abstract

Multiplex real-time PCR with quantification of targets in a single fluorescence channel has been the demand in biotechnology industry. Here, we develop a novel analytical real-time PCR technique to detect multiple targets in a single fluorescence channel without melting curve analysis. In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target. We then identify the low Tm target by utilizing a change of the fluorescence signals between two different detection temperatures. Furthermore, it enables us to determine quantification of each target in a single channel, possibly facilitating convenient patient care for drug treatment in clinics.

Evaluation of an Assay Based On Multiple Detection Temperature Technique for Simultaneous Detection of Viral Gastroenteritis-Causing Pathogens

“…These values were similar to the values (NG gDNA R 2 > 0.99) obtained in previous studies that investigated the MuDT technique using DNA targets (9), demonstrating that the one-step MuDT technique targeting RNA has a rate of performance similar to that of the viral DNA targeting. Additional investigations were conducted to demonstrate the clinical performance of this assay by comparing with a commercial kit (21).…”

“…The multiple detection temperature (MuDT) technique, introduced in 2014, is used to analyze the difference between fluorescence signals from TOCE-generated oligonucleotides amplified during the annealing and extension steps. This enables the detection of multiple targets in one channel using their individual Ct (cycle threshold) values, therefore overcoming the target number limitation (9). The MuDT technique has only been shown to be effective for DNA targets, and its applicability for the detection of RNA viruses has not been examined.…”

Clinical Performance of the Allplex^TM Respiratory Panel 1 Test Compared to Simplexa^TM Flu A/B and RSV for Detection of Influenza Virus and Respiratory Syncytial Virus Infection Including Their Subtyping

“…A single-step real-time RT-PCR was performed as described previously with minor modifications [14]. The real-time PCR primers and probes are listed in Table 1.…”

Performance Evaluation of Allplex Respiratory Panels 1, 2, and 3 for Detection of Respiratory Viruses and Influenza A Virus Subtypes

“…Recently, the Allplex respiratory panels 1, 2, and 3 (Allplex; Seegene, Republic of Korea), a one-step, real-time reverse transcription-PCR (rRT-PCR) assay based on multiple detection temperature (MuDT) technology, was developed and received Conformité Européene-in vitro diagnostic (CE-IVD) approval. The MuDT is a novel analytical technique that detects multiple targets in a single fluorescence channel without melting curve analysis (8). The assay is composed of three reaction tubes and targets 16…”